Targeted genome modification of plants has been a long-standing and elusive goal of both applied and basic research. Targeting genes and gene stacks to specific locations in the plant genome will improve the quality of transgenic events, reduce costs associated with production of transgenic events and provide new methods for making transgenic plant products such as sequential gene stacking. Overall, targeting transgenes to specific genomic sites is likely to be commercially beneficial. Significant advances have been made in the last few years towards development of methods and compositions to target and cleave genomic DNA by site specific nucleases (e.g., Zinc Finger Nucleases (ZFNs), Meganucleases, Transcription Activator-Like Effector Nucleases (TALENS) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease (CRISPR/Cas) with an engineered crRNA/tracr RNA), to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination of an exogenous donor DNA polynucleotide within a predetermined genomic locus. See, for example, U.S. Patent Publication No. 20030232410; 20050208489; 20050026157; 20050064474; and 20060188987, and International Patent Publication No. WO 2007/014275, the disclosures of which are incorporated by reference in their entireties for all purposes. U.S. Patent Publication No. 20080182332 describes use of non-canonical zinc finger nucleases (ZFNs) for targeted modification of plant genomes and U.S. Patent Publication No. 20090205083 describes ZFN-mediated targeted modification of a plant EPSPs genomic locus. Current methods for targeted insertion of exogenous DNA typically involve co-transformation of plant tissue with a donor DNA polynucleotide containing at least one transgene and a site specific nuclease (e.g., ZFN) which is designed to bind and cleave a specific genomic locus. This causes the donor DNA polynucleotide to stably insert within the cleaved genomic locus resulting in targeted gene addition at a specified genomic locus.
Unfortunately, reported and observed frequencies of targeted genomic modification indicate that targeting a genomic loci within plants is relatively inefficient. The reported inefficiency necessitates the screening of a large number of plant events to identify a specific event containing the targeted genomic loci. The screening method should also be applicable as a high throughput method for the rapid identification of plant events containing a targeted genomic loci. In addition, as targeted gene insertion occurs in conjunction with random gene insertion, screening methods must be designed to specifically identify targeting of genomic loci within a background of random insertions and to discern the genomic integration from exogenous plasmid DNA which may produce false-positive results. Furthermore, the assay should be sensitive enough to detect an event occurring in a single cell, wherein that cell contains the only targeted event amongst thousands of other non-targeted cells. Most reported plant event analyses rely on a single analytical method for confirming targeting which may lead to inaccurate estimation of targeting frequencies and low confidence outcomes. A need exists for development of improved molecular assay methods, particularly for high-throughput analysis, that can detect site specific chromosomal integrations and discern these events from exogenous plasmid DNA. Finally, current methods for assessing targeted genomic modifications are based on generation of stable plants and are time and cost intensive. Accordingly, there is a need for an analytical method that allows rapid targeting assessment at a large number of genomic loci and screening of a large number of site-specific nucleases to identify and confirm the insertion of a polynucleotide donor sequence within the targeted genomic loci.
The foregoing examples of the related art, and limitations related therewith, are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.